Endocytosis serves many important functions ranging from acquisition of extracellular nutrients to regulation of cell surface receptor expression and signal transduction, maintenance of cell polarity, and antigen presentation. Many intracellular pathogens hijack endocytic pathways in order to invade cells, proliferate, and ensure pathogen survival. Understanding the consequences of pathogenic infection has enabled greater understanding of the endocytic trafficking machinery. Isolated pathogenic genes have also been used to curb pathological processes associated with their cellular targets in human disease models. The focus of this proposal is to understand the molecular and cellular mechanisms employed by an integral membrane protein encoded by the early region 3 of human adenoviruses called RID?, which was originally identified because of its ability to divert constitutively recycling EGF receptors to lysosomes. We have recently discovered that RID? interacts with RILP and ORP1L, two known effectors for the Rab7 GTPase that governs transport from early to late endosomes and then to lysosomes. Importantly, RID? compensates for Rab7 loss-of-function, suggesting it modifies endosome membrane dynamics by coordinating recruitment of Rab7 effectors to compartments that would ordinarily be perceived as early sorting endosomes. To our knowledge this is the first protein encoded by a DNA virus that functions by Rab7 mimicry. Similar to other small GTPases, Rab7 acts by cycling between an active GTP- bound state and an inactive GDP-bound state. In contrast RID? is a non-enzymatic intrinsic membrane protein that lacks any sequence homology to Rab7, providing a remarkable example of convergent evolution. Four specific aims will test these hypotheses. 1) RID? controls microtubule-dependent vesicle transport by recruiting RILP and ORP1L which then activate minus end-directed dynein-dynactin motors. 2). RID?- RILP facilitates ESCRT-II-dependent EGF receptor sorting independent of receptor tyrosine kinase or ubiquitin status. 3) RID?-ORP1L regulates cholesterol efflux from endosomes necessary to maintain the proper lipid balance in cells. 4) RID? compensates for Rab7 loss-of-function during a productive adenovirus infection, and blunts adenovirus-induced inflammatory disease by interfering with a TNF?-EGF receptor signaling cascade that regulates IL-8 production. Altogether these studies will provide novel insights to the cell biology of membrane protein trafficking, and also the molecular basis for adenovirus-induced inflammatory disease. PUBLIC HEALTH RELEVANCE The proposed studies are relevant to public health because they will provide novel insights to the molecular pathogenesis of adenovirus pneumonia. They will generate new strategies for design of non-toxic adenovirus-based gene therapy vectors, and benefit patients with chronic obstructive pulmonary disease (COPD) and other chronic respiratory diseases where it is thought persistent adenovirus infections are an important disease risk factor. These studies will also provide the foundation for developing new treatments for patients with Niemann-Pick C disease, a progressive neurological disease that is always fatal.